In article
<Pine.SOL.4.05.10012200022450.21858-100000 at family.sogang.ac.kr>,
"Kim, Chun" <gidarim at family.sogang.ac.kr> wrote:
>> Hello.
>> I'm purifying a protein complex from fly extract. It binds well
> to DEAE-sepharose and is eluted w/ 1.5M KOAcetate, but not in
> lower salt conditions. Binding and elution wer confirmed by western
> using a specific antibody for a component in the complex. What I'm
> concerned is wheather the components of the complex are dissociated
> in such a high salt condition.
To exclude this possibility, you'd have to check the molecular weight of
your complex, either by gel chromatography (TosoHaas produces nice HPLC
gelfiltration columns, if material is precious) or by
ultracentrifugation. If you have no access to an analytical ultra, you
can use the Martin & Ames procedure (in JBC, end of the ´60s, AFAIR),
which should be precise enough for your purpose. This method is based on
centrifugation of both the protein in question and MW standards in
isokinetic glycerol- or sucrose gradients for several h. Migration
distance is proportional to MW.
Also note that dissociation of the complex need not be a problem, if it
is reversible. May be the components find together again during slow
dialysis?
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